Half-strength MS Basal Medium Research Articles

Germination of Cotyledonary Somatic Embryos into Plantlets on Coffea arabica L. of AS2K clone

Andung Sari 2 Klon (AS2K) is an Indonesian superior clone of Arabica coffee (Coffea arabica L.). Coffee plants require two years to complete their life cycle, resulting in genetic development by conventional breeding which is a significant challenge because it takes at least twenty years to produce new varieties that are ready to be marketed. Conventional coffee propagation via shoot cutting is detrimental to the strength and resistance of C. arabica L. cutting roots. Plants lack a taproot, allowing them to collapse easily, and they are frequently attacked by nematodes during the early stages of their growth. Somatic embryogenesis (SE) propagation was possible to produce an excessive number of somatic embryos for the development of plantlets during the availability of elite clones in a field experiment. The purpose of this study was to improve the plant conversion of SE by assessing the effect of different plant growth regulators (PGRs) on plantlet regeneration in AS2K clone Arabica coffee. Cotyledonary embryos were transferred to plantlet regeneration medium, which consisted of a half-strength MS basal medium containing 1 mg/L IBA supplemented with different concentrations (1.0, 3.0, 5.0 mg/l) BAP and (0.5, 1.0, 1.5 mg/l) GA3. Plantlet regeneration was established by using combination of 3 mg/L BAP and 0.5 mg/L GA3 as well as combination of 3 mg/L BAP and 1 mg/L GA3.

High throughput anther culture response in an upland rice cross ‘Khandagiri x Dular’

Aim: The experiment was conducted to maximize anther culture response in an upland indica rice cross amenable for drought tolerance breeding. Methodology: The cold pre-treated anthers of a cross Khandagiri (drought sensitive) x Dular (drought tolerant) were cultured on N6, SK3, MS and CIM media at varying concentration of hormones (2,4-D, Kn and NAA) to assess callusing response. Embryogenic calli were placed on regeneration medium (RM) with varying concentration of Kn, BAP and NAA for plant regeneration. The plantlets were acclimatized in half-strength MS basal liquid medium for one week before transferring to pot mixture (peat moss: perlite 2:1, v/v) for plant establishment in glasshouse under partial shade. Finally, the plantlets were checked for doubled haploid status by cytological study of root tips. Results: Anther culture response was found to be media and genotype-specific. F1-progenies responded better than either of the parents. A modified MS callus induction medium (CIM) with 1.5mg/l 2,4-D + 0.5mg/l Kn resulted impressively higher callusing response (30.2%) with nodular calli than SK3>MS>N6. High frequency (12.8%) of albino-free green plant regeneration with well developed rooting was achieved in RM medium (a modified MS) containing 2mg/l BAP and 0.5mg/l NAA. A total 129 doubled haploid (DH) plants (each with 12 pairs of chromosomes) were recovered which maintained normal growth, set seeds and resulted in true breeding DH lines. Interpretation: The high throughput regeneration system is amenable for doubled haploid production from indica crosses. Besides, the present doubled haploid stock can serve as an ideal mapping population and as such targeted for marker aided selection for early development of drought tolerant rice varieties.

Direct and Indirect Somatic Embryogenesis Induction in Camellia oleifera Abel.

Camellia oleifera Abel. is an important woody oil species; however, the shortage of rapid and industrialized seedling culture is a large constraint on the development of the tea oil industry. Somatic embryogenesis (SE) is one of the main powerful biotechnological tools for plant mass regeneration, but the largely unknown SE in C. oleifera limits the scale production of clonal plants. In this study, we described a high-efficiency SE system via direct and indirect pathways in C. oleifera and investigated the effect of genotype, explant age and phytohormones on SE. In the direct pathway, somatic embryos were highly induced from immature seeds 220 days after full blossom, and the development of embryoids was achieved with a combination of 0.19 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg/L thidiazuron (TDZ). In the indirect pathway, embryogenic calli were induced from the same explants in medium containing 1.5 mg/L 2,4-D, while 0.75 mg/L 2,4-D treatment led to high proliferation rates for embryogenic calli. The addition of 0.19 mg/L 2,4-D alone stimulated the production of globular embryos while causing a 75% loss of the induction rate in the heart embryo stage. Upon transfer of the globular embryos to phytohormone-free medium, an optimal induction rate of 62.37% from globular embryos to cotyledonary embryos was obtained. These data suggest that the subsequent differentiation process after the globular embryo stage in ISE is more similar to an endogenous phytohormones-driven process. Mature embryos germinated to produce intact plantlets on half-strength MS basal medium with a regeneration rate of 63.67%. Histological analysis confirmed the vascular bundle isolation of embryoids from the mother tissue. We further studied the different varieties and found that there were no significant genotype differences for SE induction efficiency in C. oleifera. Thus, we established a high-efficiency induction system for direct and indirect somatic embryogenesis (ISE) in C. oleifera and regenerated intact plantlets via SE, not organogenesis. ISE has a more complicated induction and regulatory mechanism than direct somatic embryogenesis. The improved protocol of SE would benefit mass propagation and genetic manipulation in C. oleifera.

Standardization of in vitro regeneration protocol in annual chrysanthemum

An efficient in vitro regeneration protocol of annual chrysanthemum (Chrysanthemum coronarium L.) was standardized by using leaf explants. Leaf segments (0.25 cm2) were taken from three weeks old in vitro raised plantlets and cultured on MS basal medium containing BAP, NAA and Kinetin at different concentrations. Maximum survival of micro shoots (86.67%) and shoot regeneration (82.79%) was achieved in half strength MS medium supplemented with BAP (0.5 mg/l) and NAA (1.0 mg/l) (T6). Half strength MS medium supplemented with GA3 (0.5 mg/l) (T3) showed thick shoots (5.78 cm) with dark green leaves and the highest rooting (90.00%) was recorded in the treatment including half strength MS basal medium supplemented with NAA (0.5 mg/l) (T3). The newly regenerated plantlets recorded maximum plant survival (76.60%) in medium containing cocopeat, perlite and vermiculite (1:1:1) (T2) supplemented with half strength MS inorganic broth. The study concluded that, the established protocol may be utilized in producing true to type plants which are otherwise difficult in the species. This protocol may help for large scale multiplication of desired types to fulfil the demand of quality planting material of C. coronarium.

In vitro rooting and hardening of clonal cherry rootstock Gisela 5 (Prunus cerasus × Prunus canescens)

A technique for in vitro rooting and hardening of Gisela 5 (Prunus cerasus x Prunus canescens) cherry rootstock has been developed. In vitro shoot cultures maintained by routine subculturing were used as source of microshoots for rooting experiments. In single step rooting procedure, maximum rooting (100%) was achieved on full strength MS medium fortified with 0.5 mg/l IBA with thin, long roots devoid of callus. In two step procedure of rooting, maximum rooting (60.00%) was observed after 24 hours dark incubation in half strength MS broth fortified with 0.1 and 0.4 mg/l IBA, followed by transfer to semisolid half strength MS basal medium. Addition of activated charcoal (0.4%) in half strength MS medium fortified with 0.5 mg/l IBA showed 40.06% rooting without callus, whereas in phloroglucinol (0.1%) supplemented medium only callus was observed at the bases of the shoots which did not lead to root formation. The in vitro rooted plantlets were successfully hardened in sand: soil: FYM (1:1:1) with 100 per cent survival in the month of August. After two months these plants were transferred to bigger pots containing sand: soil: FYM (1:1:1) and are showing normal growth without any morphological variations.

Callus mediated organogenesis in Lilium polyphyllum D. Don: A critically endangered Astavarga plant from North Western Indian Himalaya

Lilium polyphyllum D. Don (Liliaceae) is a critically endangered herbaceous perennial, commonly known as snow lily or Ksheerkakoli. Bulbs of the plant are of immense medicinal use and have astringent and anti-inflammatory properties. Over exploitation of the species from wild and degradation of habitats are posing threats to its existence. Therefore, an attempt has been made to develop a propagation protocol and promote mass multiplication from bulb scales of L. polyphyllum. Maximum callus was induced from the bulb scales in basal MS medium containing 2,4-D (0.5, 1, 1.5, 2 and 3 mg/l), BAP (0.5, 1, 1.5 and 2 mg/l) and NAA + BAP (0.5+2 mg/l). Maximum shooting (98.75%) was achieved in the callus when shifted to MS medium supplemented with BAP (1 and 2 mg/l) and NAA (0.5 mg/l), with an average of 1-2 cm shoot length per culture. The well developed in vitro regenerated shoots were transferred to the rooting medium and 100% rooting was achieved in half-strength MS basal medium supplemented with IBA and IAA (1 and 2 mg/l). The in vitro regenerated plantlets were shifted to a glasshouse in the sterilized soil and vermiculite mixture for acclimatization. After survival, plantlets were finally transferred to the open environment with 90% survival success.

Explant type and natural ventilation systems influence growth and content of carvacrol and thymol of Lippia gracilis Schauer

The objective of this study was to evaluate the effects of different natural ventilation systems and explant types on the growth and volatile compound content of Lippia gracilis cultured in vitro. The treatments consisted of four membrane systems (without membrane, with one, two, and four porous membranes) and two explant types (nodal segments with and without a pair of leaves). The evaluation of growth, photosynthetic pigments and chemical analysis of the volatile fraction were performed at 35 days of cultivation in half strength MS basal medium. Natural ventilation systems significantly influenced the in vitro growth and volatile fraction of L. gracilis. Explants with a pair of leaves obtained the best experimental responses. The natural ventilation system with four membranes provided the best growth parameters and leaf area response of L. gracilis explants with leaves. The photosynthetic pigments decreased with an increase in the number of porous membranes in the culture flask. Variations in the number, content, and profile of volatile compounds under the influence of natural ventilation systems were observed. Major constituents such as ρ-cymene, γ-terpinene, thymol, carvacrol, and E-caryophyllene, regardless of experimental conditions, were identified. The highest carvacrol and thymol contents were observed in plantlets grown in culture flasks with four porous membranes. To maximize the content of carvacrol and thymol from the in vitro culture of L. gracilis, explants with a pair of leaves and four porous membranes in culture flasks are recommended for use.

In vitro regeneration, production, and storage of artificial seeds in Ceropegia barnesii, an endangered plant

Approaches for in vitro regeneration and fabrication of synthetic seeds were formulated to support restoration in the wild and genetic manipulation of Ceropegia barnesii (categorized as endemic and endangered). MS medium augmented with 4 mg L−1 benzyl adenine was most advantageous for the production of multiple shoots from nodal explants. Fabrication of synthetic seeds was accomplished by sodium alginate encapsulation of nodes from microshoots. The most favorable medium combination for the induction of multiple shoots from synthetic seeds was MS medium complemented with 4 mg L−1 benzyl adenine and 1 mg L−1 gibberelic acid. Following root induction promoted by half strength MS basal medium augmented with indolebutyric acid, multiple shoots were subjected to hardening. Influence of vesicular-arbuscular mycorrhizal fungi on the hardening trials was investigated and it was observed that dual inoculation of Glomus aggregatum and G. intraradices enhanced the survival rate. The encapsulated nodes of C. barnesii were tested for their capability to endure different temperatures during storage and the optimal temperature for storage was found to be 4°C. A methodology for initiation of somatic embryogenesis from C. barnesii is also reported here, but embryos could not be induced to develop further. The micropropagated plants were reintroduced in to their natural habitat. This is the first report on micropropagation of C. barnesii.

Tissue culture studies and estimation of camptothecin from Ophiorrhiza prostrata D. Don

Camptothecin is an important molecule in the synthesis of some of major anticancer agents such as irinotecan and topotecan. Traditional sources of camptothecin are mainly woody plants, some of which are being overexploited to a level that threatens the existence of the respective species. Alternate sources are being identified. In the current study, a tissue culture protocol was developed for the mass multiplication of Ophiorrhiza prostrata, an herbaceous camptothecin-containing plant, using seed explants, in order to evaluate its potential as an alternate source of commercial camptothecin. The germination of seeds was standardized on half-strength solid basal MS medium (94.8 ± 2.65% success rate). Seedlings subcultured on half-strength MS agar medium supplemented with 1.5 mg l−1 benzylaminopurine (BAP) produced the most shoots (28.0 ± 2.58) from the nodal region of a single seedling. Mass multiplication of in vitro shoots was achieved by subculturing a single shoot as well as shoot clumps. Subculturing shoot clumps containing a minimum of 4–6 shoots yielded fewer shoots per node (32.2 ± 2.03) than when single shoots were subcultured (25.0 ± 2.0). Shoot elongation was achieved on half-strength MS basal medium in a period of 2–3 weeks. Various concentrations of auxins were tested for rooting, and 1 mg l−1 NAA gave the highest number of roots (44.0 ± 4.13). Rooted shoots were successfully established under field conditions. High-performance thin-layer chromatography (HPTLC) was employed to measure the camptothecin levels in wild-grown plants (1.46 ± 0.02 µg g−1 dry wt), in-vitro-regenerated plants (1.10 ± 0.02 µg g−1 dry wt), and in-vitro-derived hardened plants (1.83 ± 0.02 µg g−1 dry wt). The results of this study indicate that in vitro methods could be a potential path to the rapid expansion of plant biomass, which could serve as a potential source of commercial camptothecin.

An Efficient Protocol Of Stevia rebaudiana Regeneration For Large-Scale Production

Stevia rebaudiana is considered a valuable natural sweetener and medicinal plant containing sweetener compounds in its leaves. Nowadays, plant drugs are frequently considered to be free from side effects and less toxic than the synthetic one, for that people and some pharmacological companies are heading to the herbal medicine. Thus, to meet the increased demand for raw material of this medicinal herb, I described in this study an efficient regeneration protocol using shoot tip and nodal explants and different PGRs on MS medium for healthy biomass production. The optimum shoot growth with a high number without callus formation was obtained using MS medium in the presence of 1.0 mgL-1 BA after 6 weeks which shows (4.06-4.46) shoot buds/explant after 6 weeks. The combination of 1.0 mg L-1 BA with 0.5 mg L-1 IBA was found to be best combination for shoot multiplication that recorded 6.66 shoots/explant with 4.46 cm shoot length. Healthy elongated shoots were harvested and then cultured on half strength MS basal salts medium without vitamins medium in the presence of different concentrations of IBA and NAA individual or in combination with IAA or 2,4-D. Moreover, the optimum root growth with high roots number (4.46) with (76.6 %) root formation was observed using 1.0 mg L-1 IBA on half strength MS medium. Therefore, well-developed healthy in vitro rooted plantlets after 6 weeks were successfully acclimatized and they grew as normal plants in the greenhouse.

Establishment of in vitro propagation protocol for Hybrid Tea rose cv. Raktagandha

An efficient protocol for in vitro multiplication of hybrid tea rose cv. Raktagandha was developed using axillary bud segments. Out of different pre-treatments of explants tried, the highest explant survival (78.05%) was obtained with carbendazim (0.2%) + diathane M-45 (0.2%) + 8-HQC (200 mg/l) for 3 h on a horizontal shaker (120 rpm). Sucrose concentration of 30 g/l in the medium seems to be optimal for in vitro shoot multiplication. Murashige and Skoog medium supplemented with 3.5 mg/l BAP + 0.2 mg/l NAA + 0.5 mg/l GA3 was found most effective for culture establishment and shoot proliferation with highest number of micro-shoots (4.62 shoots/explant). Rooting of micro-shoots was induced on half-strength MS basal medium supplemented with NAA (0.5 mg/l) + IBA (0.5 mg/l). The regenerated plantlets were efficiently hardened in glass jars filled with vermiculite + agropeat (1:2) moistened with half-strength MS medium salts and covered with polypropylene lids, thereafter plants were successfully transferred to the glasshouse with good survival.

In vitro plant regeneration, flowering and fruiting from nodal explants of Andrographis lineata nees (Acanthaceae)

In the present study, in vitro propagation of tribal endemic medicinal plant, Andrographis lineata has been established using mature nodal explants. High frequency of regeneration (91.4%) was achieved on MS medium containing BA (3.0 mg L–1) along with IAA (0.2 mg L–1). Strikingly, irrespective of the season and collection period, we observed axillary flower induction and fruit formation from the above in vitro cultures supplemented with BA and NAA. Transition from the vegetative to the reproductive phase occurred within 2 months of culture, and importantly was influenced by factors such as sucrose and cytokinins. In vitro-regenerated flowers were morphologically identical to in vivo flowers. Furthermore, our scanning electron microscopic studies revealed that pollen external morphology of both in vitro and in vivo flowers were similar. The flowers self-fertilized and produced fruits in vitro. Elongated shoots rooted well on half-strength MS basal medium, and were successfully acclimatized to the garden conditions. Altogether, our established protocol can be utilized in plant breeding for the purpose of ex situ conservation, quick flowering, and fruit set.

Cost Effective Techniques for In Vitro Propagation of Two Varieties of Sugarcane

The present study was carried out to optimize cost effective in vitro protocol for mass propagation of two commercial sugarcane varieties grown in Maharashtra, India. The effect of different cytokinins and auxins on in vitro initiation, multiplication and rooting was investigated in sugarcane (Saccharum spp.) varieties; Co86032 and CoC671. Shoot tip explants comprising of apical meristems and 1-2 leaf primordia were carefully excised from the apical region and inoculated on MS basal liquid medium supplemented with different concentrations and combinations of Kinetin and BAP. Only initiation medium was sterilized by autoclaving and further shoot multiplication, rooting was done on medium sterilized with sodium hypochlorite (4% w/v available chlorine) at the concentrations 0.05% corresponding to total active chlorine concentration 0.002% in the medium. With regard to shoot initiation, variety Co86032 showed maximum shoot formation (96.67%) on MS medium supplemented with BAP (0.2mg/L) and Kinetin (0.1mg/L) with 3% sucrose and 10% CW on the other hand variety CoC671 showed maximum shoot formation (93.33%) on MS medium supplemented with only Kinetin( 0.5mg/L), 3% sucrose, 10% CW. The best response in terms of shoot multiplication for variety Co86032 was observed on MS medium with BAP (0.2mg/L) and Kinetin (0.1mg/L)with 3% commercial sugar while for variety CoC671 it was observed on MS medium with Kinetin and BAP (0.2mg/L each) and 3.0% commercial sugar. Variety Co86032 produced multiple shoots with an average number of 31.25±0.99 shoots per bottle while variety CoC671 produced 27.72±0.66 shoots per bottle. For rooting, the multiplying shoots were separated in smaller groups and transferred on half strength MS liquid medium containing different concentrations of NAA or IBA with different concentrations of commercial sugar. Half strength MS basal medium with NAA (5.0 mg/L) and 5.0% commercial sugar induced highest rooting (84.44%) with average root number per shoot of 8.49 ±0.25 in Co86032. In variety CoC671, half strength MS basal medium with IBA (5.0 mg/L) and 5.0% commercial sugar induced highest rooting (82.22%) with average root number per shoot of 6.72 ±0.48. The use of commercial sugar can effectively replace the use of expensive sucrose in in vitro multiplication of sugarcane varieties. All culture media were used in liquid state instead of semisolid agar media. Rooted shoots were transplanted in green house for hardening. Use of liquid media, chemical sterilant Sodium hypochlorite (NaOCl) and commercial sugar helped in reducing the cost per plantlet to a considerable extent.

An efficient protocol for in vitro propagation of Fig (Ficus carica sp) and evaluation of genetic fidelity using RAPD and ISSR markers

Fig had been proliferated in vitro using tissue culture techniques. However, factors affecting shoot proliferation have not been optimized. In the present study, sufficient protocol for in vitro propagation of fig trees (Ficus carica sp) using shoot tips and nodal explants were reported. High percentage of shoots proliferation (95%) was obtained by culture of nodal explants containing two buds that were excised from greenhouse plants on MS medium supplemented with 3.0 mg/l BAP and 0.1mg/l Kin. Multiple shoots (12 multiple shoots per explant) were obtained after subculture of formed shoots on MS medium supplemented with 2.0 mg/l BAP, 0.1 mg/l Kin and 0.1 mg/l gibberellic acid (GA3). Subsequently, high frequency of rooting (100%) were obtained after transferring the plantlets to half strength MS basal medium supplemented with 2.0 mg/l indole-3-Acetic acid, 0.1mg/l Indole butyric acid and 2g/ l activated charcoal. Rooted plantlets were planted in pots containing a sterile soil and kept in the green house; the acclimatization was performed successfully with 100% survival rate. One year old plants were exhibited normal morphological characters comparing with the mother plant. Genetic homogeneity of the micropropagated plants was evaluated by molecular analysis using nine randomly selected 1-year-old fig plants along with the mother plant. A total of six RAPD primers and 5 inter-simple sequence repeat (ISSR) primers produced a total of 109 resolvable, reproducible and scorable bands ranging from 250 to 1550 bp in size. Among these bands, 103 bands were monomorphic (94.5%) and 6 bands were polymorphic (5.5%). This low polymorphism ration between mother plants and micropropageted plants indicates the little effect of somaclonal variations, the high genetic similarity between mother plants and micropropageted plants and demonstrates the reliability of our in vitro propagation system for fig trees.

Germinação in vitro e aclimatização de Hamatocactus setispinus

Seed propagation preserves the population genetic variability and helps selecting desirable features. This study evaluated the in vitro germination of Hamatocactus setispinus in six different culture media, 1- MS basal medium full strength; 2- half-strength MS basal medium; 3- 1.0 g L-1 of Peter’s CalMag® 15-05-15 formulation; 4- 0.5 g L-1 of Peter’s CalMag® 15-05-15 formulation; 5- MS basal medium supplemented with 10% coconut water and; 6- water and agar, with and without activated charcoal, and the speed of germination index, the mean germination time and the germination rate, root length, shoot length and the number of roots were evaluate. The seedlings with superior development obtained from in vitro germination were acclimatized in two substrates: Biomix® Floreira; Biomix® Floreira + sand. Seedling survival, shoot length, shoot diameter, root length, root number, shoot fresh matter weight, root fresh matter weight, shoot dry matter weight and root dry matter weight were evaluated. Peter’s 1.0 g L-1 medium without activated charcoal led to the best results for root length (11.36 mm) and root number (3.84). There was 100% of seedling survival. Acclimatization substrates did not differ among themselves and, therefore, they did not affect seedling growth.

In Vitro Seeds Germination and Seedling Growth of Bambara Groundnut (Vigna subterranea (L.) Verdc. (Fabaceae)).

Bambara groundnut (Vigna subterranea (L.) Verdc.) is an indigenous grain legume. It occupies a prominent place in the strategies to ensure food security in sub-Saharan Africa. Development of an efficient in vitro regeneration system, a prerequisite for genetic transformation application, requires the establishment of optimal conditions for seeds germination and plantlets development. Three types of seeds were inoculated on different basal media devoid of growth regulators. Various strengths of the medium of choice and the type and concentration of carbon source were also investigated. Responses to germination varied with the type of seed. Embryonic axis (EA) followed by seeds without coat (SWtC) germinated rapidly and expressed a high rate of germination. The growth performances of plantlets varied with the basal medium composition and the seeds type. The optimal growth performances of plants were displayed on half strength MS basal medium with SWtC and EA as source of seeds. Addition of 3% sucrose in the culture medium was more suitable for a maximum growth of plantlets derived from EA.

Genetic fidelity of long-term micropropagated Lavandula officinalis Chaix.: an important aromatic medicinal plant

A protocol for long-term in vitro conservation of multiple shoot cultures of Lavandula officinalis and regeneration of true-to-type plants from them is described here. Multiple shoots were developed from apical bud explants on a modified vitamin-enriched Murashige and Skoog’s basal medium supplemented with 2.5 mg/l Kinetin. The cultures were subsequently conserved in vitro for 6 years under slow growth conditions imposed by lowering the sucrose level from 3 to 2.0 % and increasing the agar concentration from 0.8 to 1.2 % in this medium. Complete plant regeneration from in vitro conserved shoots was achieved through axillary shoot proliferation on original 2.5 mg/l Kinetin containing medium followed by rooting of individual shoots in a hormone-free half strength MS basal medium. The genetic fidelity of the regenerated plants was tested by random amplified polymorphic DNA analysis. Twenty one arbitrary decamer primers produced a total of 64 scorable bands (1–6 bands/primer) in the size range of 100–5,148 bp. All DNA fingerprints with these primers displayed monomorphic band profiles indicating homogeneity among the regenerated progeny and their uniformity with the donor parent. Head-space gas chromatographic analysis of the leaf tissue excised from mother as well as regenerated plants also revealed a similar qualitative and quantitative profile of volatile terpenes. The utility of the developed protocol for long-term ex situ conservation and quality plant production for cultivation of this high value aromatic herb is discussed.

In vitro propagation and synseed production of scarlet salvia (Salvia splendens)

The present study provides a protocol for high-frequency in vitro propagation and short-term conservation using encapsulation technology of Salvia splendens. Nodal segments were more responsive than shoot tips in terms of shoot multiplication. Murashige and Skoog’s (MS) basal medium augmented with 5.0 µM 6-benzyl adenine (BA) was optimum for shoot induction through both explants. MS medium supplemented with 5.0 µM BA and 0.5 µM α-naphthalene acetic acid induced a maximum of 15.0 shoots per nodal segment and 5.0 cm shoot length after 4 weeks of incubation. Microshoots were best rooted (30.6 roots per shoot) on half-strength MS basal medium containing 2.5 µM indole-3-acetic acid (IAA). For synseed preparation, nodal segments were encapsulated in 4 % Na-alginate with different gel matrices and 100 mM CaCl2·2H2O (complexing agent). Maximum conversion (63.6 %) of synseeds into plantlets was achieved on MS basal medium when they were prepared with the gel matrix of MS basal medium supplemented with 5.0 µM BA and 2.5 µM IAA. Pretreatment of synseeds with 100 mM KNO3 for 30 min, prior to incubation on the planting media significantly improved conversion frequency (94.6 %). Synseeds could be stored up to 10 weeks at low temperature (4 °C). Complete plantlets were successfully acclimatized and established in field.

Biodiversity and In vitro Conservation of Three Medicinally Important Herbs: Spilanthes acmella L. var. oleraceae Clarke, S. calva L., and S. paniculata Wall. ex DC.

In vitro mass production and conservation of three antimalarial medicinal herbs: Spilanthes acmella L. var. oleraceae, S. calva, and S. paniculata of asteraceae employing seedling leaf explants. The explants were reared on MS medium containing a variety of growth regulators, namely cytokinins (BA, KN and 2iP), or auxins (2,4-D, NAA and IBA) in different concentrations (0.1, 1, 5, 10, and 20 μM) either alone or in combination. MS+5μM BA proved optimum for S. calva, with an average of 4.17 ± 1.5 shoots. However, in S. acmella L. var. oleraceae and S. paniculata, MS+1μM NAA+5μM BA proved best for differentiating an average of 4.46 ± 1.2 and 6.50 ± 2.6 shoots, respectively. The shoots were excised and rooted in half-strength MS basal medium supplemented with auxins (NAA or IBA). Thus, a successful method for ex-situ conservation has been established in the three Spilanthes species for the first time.

Efficient plant regeneration, genetic fidelity and high-level accumulation of two pharmaceutical compounds in regenerated plants of Valeriana officinalis L.

An efficient regeneration system via adventitious shoot formation was developed in Valeriana officinalis, an important medicinal species. Regeneration response was significantly influenced by the type and concentrations of plant growth regulators as well as callus morphology. Optimum values for adventitious shoot–buds induction in both leaf and petiole explants were obtained when organogenic calli with compact, nodular and yellowish green phenotype was cultured in MS medium supplemented with 9.30μM Kn and 2.26μM 2,4-D. Well-developed shoots was achieved on a hormone-free half strength (1/2) MS basal medium. In vitro rooting of elongated shoots was tried on 1/2 MS medium supplemented with various concentrations of α-naphthalene acetic acid (NAA) or indole-3-butyric acid (IBA). IBA at a concentration of 2.46μM was found to be the best rooting treatment, which promoted the highest frequency of rooting (98%). Successful acclimatization of rooted plantlets was achieved in potting medium containing 5:1 mixture of peat and perlite with 95.34% survival rate. The contents of valtrate and valerenic acid in biomass extracts from petiole-derived plants were significantly (P<0.05) higher than leaf-derived plants as well as the seed-raised field-grown plants. Maximum valtrate (6.98mgg−1 DW) and valerenic acids (3.02mgg−1 DW) contents were quantified in root tissue of petiole-derived plants raised on medium with higher concentration of Kn (9.30μM) in combination with 2.26μM 2,4-D. Random amplified polymorphic DNA (RAPD) analysis indicated no evidence of genetic variation in the tissue culture-raised plants. The results suggest that the tissue culture-raised V. officinalis plants are capable of producing higher content of secondary metabolites under optimum in vitro conditions than that under natural wild growth conditions.